As CD4+ T cells are important not only for the development but also for the persistence of the anti-tumor immune responses, we have hypothesized that the failure to stimulate this CD4+ T cells may be one of the reasons why previous peptide vaccines did not induce tumor regression in a large number patients with melanoma. We have recently identified a series of MHC class II-presented melanoma epitopes recognized by CD4+ T cells. In particular, we have identified several epitopes derived from the NY-ESO-1 antigen that is commonly expressed by a broad range of tumor types, including melanomas. We now propose to perform clinical trials to assess the in vivo efficacy of cancer vaccines with T-helper epitopes in patients with melanoma. The rationale for our proposed research project is several fold and can be stated as follows:(1) vaccines integrating T-helper epitopes will optimally stimulate high-avidity tumor-reactive CTL responses capable of mediating tumor regression; (2) T-helper and CTL responses against epitopes that derive from NY-ESO-1 are less likely to be tolerized in situ since this antigen is not expressed by normal tissues; (3) CpG oligonucleotide adjuvants are expected to stimulate plasmacytoid dendritic cells (PDCs) resulting in strong Th1-type immunity; (4) modern immunologic monitoring techniques will allow us to discriminate melanoma-specific T cell responses in the blood of vaccinated patients, allowing us to assess the in vivo impact of our vaccine strategy. We propose the following aims: (1) To perform a phase I-II randomized clinical trial of NY-ESO-1-derived peptides versus recombinant NY-ESO-1 protein in patients with melanoma, using CpG 7909 as adjuvant; (2) To analyze the epitope-specificities and functions of CD8+ and CD4+ T cells from the blood of the patients undergoing peptide or protein-based vaccines in the Specific Aim 1; (3) To study the trafficking and immunostimulatory effects of plasmacytoid dendritic cells (PDCs) following vaccination with CpG + peptides or protein. While we cannot predict the results of the clinical trial proposed in Aim 1, those results should facilitate a better understanding of the mechanisms underlying peptide-based and protein-based vaccination through skin in association with CpG 7909 for patients with melanoma. Altogether, these data will indicate whether or not one vaccination intervention in Aim 1 is superior to the others in promoting anti-NY-ESO-1 CTL responses. These data will also allow us to define additional strategies that may synergize with CpG adjuvants to further improve vaccines designed to enhance the development and durability of tumor-reactive CD8+ T cells and Th-1 type CD4+ T cells.